Automatic plasma separation and metering

ABSTRACT

A new method is disclosed for extracting plasma from whose blood and metering the amount of plasma to an exact volume for dispensing into a diagnostic test, in a fully automatic and self-contained device. The device can be used in resource limited settings by unskilled users to facilitate sophisticated medical diagnostic testing outside of a hospital, clinic or laboratory.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of U.S. applicationSer. No. 17/163,711, filed on Feb. 1, 2021, now U.S. Pat. No.11,717,827, which claims priority to and the benefit of U.S. applicationSer. No. 15/745,707, filed on Jan. 18, 2018, now U.S. Pat. No.10,906,042, which is a national-stage application filed under 35 U.S.C.371 of PCT Application No. PCT/US2016/042865, filed on Jul. 18, 2016,which claims priority to and the benefit of U.S. provisional applicationNo. 62/195,281, filed on Jul. 21, 2015 and U.S. provisional applicationNo. 62/249,922, filed on Nov. 2, 2015, which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Field of the Invention: This invention discloses a method of extractingplasma from whole blood in an automatic fashion using a plasmaseparation membrane, capillary forces, and a novel capillary reset valveor mechanism (CPR-Valve). The CPR-Valve automatically overcomes thebreakthrough pressure of the plasma separation membrane allowing theplasma extraction, collection and metering processes all to take placewithout any external forces, energy or action on the part of the user orneed of additional support equipment.

Description of Related Art: Many attempts have been made at separatingplasma from whole blood in a stand-alone device, as a means of replacingthe common method of centrifugation. Plasma is often required for thedetection of blood-based biomarkers because, in many cases, the cellularcomponents of blood interfere with detection of these biomarkers.Centrifugation, however, is a fairly complex task involving skilledpersonnel, complex, heavy and large equipment, and electrical power. Inaddition, a means for obtaining the whole blood is needed, which isusually performed by a venous puncture and blood draw by another skilledperson, namely a phlebotomist. Finally, another or one of the sameskilled workers is needed for performing the centrifugation, measuringthe required volume of plasma, and dispensing the plasma into thediagnostic device, usually using a pipette. In automated systems some ofthese manual steps can be replaced with robotic processes of loading,spinning, measuring and dispensing. However, these automated systems areusually even more complex, expensive, large and sophisticated than asimple centrifuge. The need for replacing all of these steps in asimple, affordable, and easy to use device is the focus of numerous, asyet commercially unsuccessful, efforts.

Many attempts at replacing the centrifuge use commercially availablePlasma Separation Membranes (PSM), such as the Pall Vivid™ PSM orInternational Point of Care Primecare PSM. Numerous scientific articleshave been published describing devices comprised of these membranes, aswell as support structures required for adding the whole blood,collecting the separated plasma and, in all cases, some means ofovercoming the breakthrough pressure of the PSM to allow the plasma topass through the membrane and be collected.

Many of these articles detail the biochemical nature of the plasma thatis collected and compare it to plasma derived from centrifugation. Mostcomparisons are favorable, which should lead to rapid adoption of theseprocesses due to the commercial need of such technology. However, it hasnot, and there is, to date, no readily available and commonly usedcentrifuge replacement device. This is most likely due to the complexmethod of sample handling, collection, difficult, complex, or expensivemethod of overcoming the breakthrough pressure of the PSM, and relatedissues that lead to designs that are not commercially viable, ether dueto cost, complexity of use, or poor performance.

BRIEF SUMMARY OF THE INVENTION

The disclosed technology details a new method of overcoming thebreakthrough pressure of hydrophilic membranes, in this case the PlasmaSeparation Membrane (PSM), and the design advantages made possible bythis new method. These design advantages include the ability to designexactly where the plasma breaks through, how the flow of plasma throughthe membrane proceeds, where it stops, and how all parameters can beeasily controlled using passive capillary forces, leading to acompletely automatic, self-contained, disposable device that is easilyused and inexpensive to manufacture.

A device for overcoming the breakthrough pressure of the PSM makes useof a new mechanism described as a Capillary Pressure Reset (CPR) Valveor mechanism. This valve is comprised of a soluble matrix that has ahigh enough capillarity to draw liquid through a hydrophilic membrane orfilter (e.g., the PSM) under passive capillary forces, therebyeliminating the membrane's inherent breakthrough pressure, and thendissolving in the extracted liquid (the plasma) and releasing the liquidinto a new geometry that has lower capillarity.

By placing the soluble matrix comprising the CPR-Valve in a strategiclocation at one end of a plasma pooling or collection channel, theinitial breakthrough point and direction of flow of the plasma can bedesign to proceed in a repeatable manner. This flow or filling processcan be designed to ensure no bubbles are trapped, which can lead toaccurate, repeatable filling of a precise geometry of known volume,leading to the plasma being metered to a known volume prior todispensing.

Several examples of how the technology is used, and specific details ofthe design parameters of complete systems, are disclosed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The following drawings illustrate exemplary embodiments for carrying outthe invention. There are, in fact, many possible configurations,housings, flow systems, entrance and exit point designs, flow patterns,Capillary Pressure Reset (CPR)-Valve placements, dimensions andgeometries including rectangular or cylindrical flow channels, andliquid flow driving forces possible in various embodiments of theinvention. The following examples only serve to illustrate principlesdiscussed in this disclosure, and are not meant to be limiting in anyway in converting the principles discussed in this disclosure intophysical form, and are not necessarily to scale as may be used in aphysical system. Like reference numerals refer to like parts indifferent views or embodiments of the present invention in the drawings.

FIG. 1 illustrates the basic concept of CPR-Valve placement in contactwith the bottom surface of a Plasma Separation Membrane (PSM), and theflow of plasma to fill a space under the PSM.

FIGS. 2 a and b illustrates assembled (a) and exploded (b) views of astructure useful for separating plasma from a whole blood sample anddelivering a metered volume of that plasma into a capillary tubeinserted into the plasma pooling area according to an embodiment of theinvention.

FIG. 3 illustrates a layer by layer view of a thermoformed orinjection-molded structure useful for separating plasma from a wholeblood sample introduced into a metering input capillary and the plasmacollected into a specially modified capillary tube designed to alsofunction as a removable pipetting device according to an embodiment ofthe invention.

FIG. 4 illustrates a similar design as FIG. 3 , except that this designis comprised of laminated layers rather than injection molded orthermoformed parts according to an embodiment of the invention.

FIG. 5 illustrates another laminated plasma extraction and meteringdevice where the metered plasma is dispensed directly from the body ofthe device, rather than collected into a removable capillary pipetteaccording to an embodiment of the invention.

FIG. 6 illustrates an alternative geometry of a plasma extraction andmetering device where the separated plasma is collected into tworemovable capillary pipettes useful for aliquoting the whole blood inputinto two separate plasma outputs for dispensing into two differentdiagnostic assays according to an embodiment of the invention.

FIG. 7 illustrates a multi-layered laminate design with a short and deepplasma collection channel and no vent. In this design metering isentirely controlled by the output capillary pipette according to anembodiment of the invention.

FIG. 8 illustrates a design where the CPR-Valve serves a dual purpose,namely to draw plasma through the PSM and to overcome the capillary stopjunction at the entrance of the large inner-diameter capillary pipetteby drawing plasma into the capillary pipette, according to an embodimentof the invention.

FIG. 9 illustrates further a profile view of the CPR-Valve and itsplacement between the PSM and output capillary pipette in order toperform the dual purpose mentioned in connection with FIG. 8 , accordingto an embodiment of the invention.

FIG. 10 illustrates a profile view of the CPR-Valve and its placementbetween the PSM and a reagent pellet or disk and the output capillarypipette according to an embodiment of the invention.

FIG. 11 illustrates a profile view of a multi-layered laminate designshowing an extension of the plasma pooling area useful for improving theefficiency of a device according to an embodiment of the invention.

FIGS. 12 a and 12 b illustrate the use of a capillary stop junction nearthe outlet of the input capillary to ensure precise volume control ofthe input whole blood added to the PSM according to an embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Certain embodiments as disclosed herein provide for an automatic plasmaseparation device. After reading this description it will becomeapparent to one skilled in the art how to implement the invention invarious alternative embodiments and alternative applications. Althoughvarious embodiments of the present invention will be described herein,it is understood that these embodiments are presented by way of exampleonly, and not limitation. As such, this detailed description of variousalternative embodiments should not be construed to limit the scope orbreadth of the present invention.

Capillary Pressure Reset (CPR)-Valve: The breakthrough pressure of amembrane is defined as the pressure required to force liquid (filtrate)through the downstream surface of a membrane. In simplified form thebreakthrough pressure can be considered as multiple capillary stopjunctions as the pores on the bottom surface of the membrane open to thespace below the membrane. The breakthrough pressure is dependent on themembrane material composition, pore size and liquid filtrate properties.In the disclosed design the soluble matrix of which the CPR-Valve iscomprised is in physical contact with the bottom surface of the PlasmaSeparation Membrane (PSM) and represents a zone of higher capillaritythan the PSM itself. The CPR-Valve material is usually comprised of asimple sugar, but can take many forms depending on the needs of thesystem. The placement of the CPR-Valve in physical contact with thebottom surface of the PSM causes the liquid (plasma) within the PSM toflow into the CPR-Valve under capillary action. Once plasma is withinthe CPR-Valve, the soluble material dissolves, releasing the plasma as afree-standing drop into the space between the PSM and the base of theplasma pooling area. In this fashion, no external pressure is applied inorder for the PSM's inherent breakthrough pressure to be overcome. Thedroplet of plasma forms a connection or meniscus between the bottomsurface of the PSM and the base of the pooling area. The presence ofplasma within this space and in contact with the bottom surface of thePSM allows the plasma to continue to flow through the bottom surface ofthe PSM as it wicks along the space, with the meniscus being drawnforward due to capillary action, eliminating the capillary barriersretaining additional plasma within the small bottom pores of the PSM.This contributes to additional flow of plasma into the pooling area andcauses the meniscus to spread further. The capillary force caused by themeniscus bound by the hydrophilic surfaces of the bottom surface of thePSM and base material continues to draw meniscus forward, breakingadditional capillary barriers as it spreads. In this manner the entiresurface area of the PSM can be recruited to separate plasma, not just inthe area of the placement of the CPR-Valve.

FIG. 1 illustrates a typical geometry profile of the PSM 100, CPR-Valve200 and base material 310. The spread or movement of the meniscus 400formed between the PSM and base material leading to a point 420 wherethe separated plasma may be collected. FIG. 2 illustrates how acapillary tube 600 can be introduced into the plasma pooling space 410through a hole 450 in the bottom of the base material 310. Plasma willflow into the capillary by capillary force provided the physical andmaterial parameters of the capillary tube satisfy the conditions ofcontinued capillary flow, to be discussed more later in this disclosure.

Flow through the PSM will continue until all plasma available forcollection has passed through, until the pores of the PSM become blockedby cellular components of the whole blood or until the lagging end offlow generates a higher capillary barrier than is present at the leadingend of flow. However, if flow does not continue through the PSM, it isstill possible to collect, or extract, all plasma that has pooled underthe PSM. This is possible by placing a venting duct 330 in the system,FIG. 2 , which allows air to pass into the plasma pooling space 410 asplasma is drawn out of the same space and into the capillary tube 600.The vent 330 acts as a capillary barrier and does not allow plasma toexit the plasma pooling area 410 under normal operating conditions. Lackof venting would generate a vacuum back pressure, which would preventany additional plasma from being collected, except what may be pulledthrough the PSM by the capillary forces of the capillary itself, inexcess of the volume of the plasma pooling area.

The strategic placement of the vent 330 will also allow the region underthe PSM, where the original CPR-Valve 200 was located, to be bypassedwhile plasma is being collected from the plasma pooling area 410. Thiscauses all, or the majority, of dissolved soluble matrix of which theCPR-Valve is comprised, to remain in the plasma pooling area and not bepassed into the capillary tube 600.

By strategic placement of the CPR-Valve, vent, and capillary output, asystem can be produced that reliably and repeatedly allows for plasma tobe extracted from whole blood, fill a pre-defined space or channel,defined by the geometry of the space walls 320, top 100 and base 310,without trapping bubbles, and deliver the extracted plasma into acapillary tube 600, which can be removed and used to dispense thecollected plasma anywhere it is needed.

Capillary Pipette: The physical and material parameters that are neededfor plasma to enter the inserted capillary tube under passive capillaryforces are defined by the capillary force equation shown here:

$\begin{matrix}{P = {- \frac{2\sigma\cos\theta}{r}}} & (1)\end{matrix}$

-   -   where: P Capillary Pressure    -   r radius of pore or flow channel    -   θ contact angle of channel or membrane material    -   σ surface tension of liquid

A negative pressure is similar to a suction force that draws liquid intothe system. A contact angle greater than 90° represents a hydrophobicmaterial, and the resulting capillary pressure turns positive,indicating a positive force is required to push liquid into the system.

At the point where the capillary tube enters the plasma pooling area, aninterface exists between two different geometries. In order for plasmato flow into the capillary tube, the capillary pressure, or capillarity,of the geometry and material of the capillary tube must be greater thanthe capillarity of the geometry and materials comprising the plasmapooling area. In other words, the difference between P_(cap) (or justP_(c)) and P_(pool) (or just P_(p)) must remain negative, as isillustrated by this equation:

$\begin{matrix}{{\Delta P} = {{{P_{c}\left( {\sigma_{c},\theta_{c},r_{c}} \right)} - {P_{p}\left( {\sigma_{p},\theta_{p},r_{p}} \right)}} = {\frac{2\sigma_{p}\cos\theta_{p}}{r_{p}} - \frac{2\sigma_{c}\cos\theta_{c}}{r_{c}}}}} & (2)\end{matrix}$

The surface tension (σ) between the liquid (plasma) and air does notchange from one geometry to the next, so σ_(c)=σ_(p), which allows theequation to be simplified to:

$\begin{matrix}{{\Delta P/2\sigma} = {\frac{\cos\theta_{p}}{r_{p}} - \frac{\cos\theta_{c}}{r_{c}}}} & (3)\end{matrix}$

The geometry of the flow channel in the capillary tube is cylindrical,with a known radius r_(c), but the geometry of the pooling area isrectangular, requiring an effective hydraulic radius to be calculated,based on the equation:

$\begin{matrix}{r_{p} = \frac{ab}{a + b}} & (4)\end{matrix}$

-   -   where: r_(p) Effective hydraulic radius of the pooling area    -   a height of pooling area flow channel    -   b width of pooling area flow channel

For example, in a case, such as where the height of the pooling areaflow channel is 170 μm, and the width is 1250 μm, the effective radiuscan be calculated to be approximately 150 μm. In the case where thecapillary tube also has a radius of approximately 150 μm, equation (3)can be simplified to:

ΔPr/2σ=cos θ_(p)−cos θ_(c)  (5)

If the material of the pooling area is mainly derived of hydrophilicplastics, which may have a contact angle (θ_(p)) of around 75°, and thecapillary tube is comprised of glass, with a contact angle (θ_(c)) ofaround 15°, cos(75°)=0.26 and cos(15°)=0.97, the difference is negative,allowing flow to continue into the capillary tube.

In the case where the radii of the two geometries are not approximatelythe same, and it is desired to derive an equation that clarifies whatthe relationship between r_(c) and r_(p) should be, in order for thecapillary pressure at the junction to remain negative, the followingrelationship can be derived from equation (3), namely:

r _(p) cos θ_(c) >r _(c) cos θ_(p)  (6)

Using the same contact angles as before, and rearranging to emphasizethe desired radius of the capillary tube, we obtain:

r _(c)<3.7r _(p)  (7)

While this relationship is useful to keep in mind, it does not take intoaccount some very critical issues associated with microfluidics,capillary forces, and commercially viable manufacturing and assemblyissues in general. These issues include variability in materialproperties, surface roughness, and in particular, precise alignmentbetween surfaces and junctions.

Considering the illustration shown in FIG. 3 , an input meteringcapillary 500 is used to deliver a minimum volume of whole blood to thePSM 100. A cover 550 is also used to facilitate wicking of the bloodacross the whole PSM, rather than just pooling nearest to the inputcapillary. The alignment between the input capillary 500, the cover 550,and PSM 100 is important to ensure blood reaches the PSM and can bedistributed. The blood in the capillary will also be subject to gravity,in addition to capillary forces, such that, if the capillary tube doesnot fully reach the PSM, the blood in the capillary tube may extendslightly past the end of the capillary tube to aid in bridging any gap.

However, if the capillary tube extends too far past the bottom surfaceof the cover, it may become imbedded into the PSM, preventing properwetting of the PSM and adequate pooling of blood across the wholesurface area of the PSM to facilitate even plasma extraction.

Also to facilitate even distribution of the input blood sample, an inputvent 510 is needed. As blood covers the PSM, air in the space betweenthe PSM and the cover and air within the PSM must be vented out. Thismay take place by air passing through the plasma pooling area 410 andout through the output capillary pipette 650, but it is more reliable tohave a separate vent 510, either in the cover, as is illustrated in FIG.3 , or in the base, as is illustrated in FIG. 4 . In either case, it isuseful for the vent to be placed in a position where blood or plasmadoes not leak out during normal device operation. Also, as isillustrated in FIG. 4 and again in FIG. 7 , it may be useful for thevent to be offset slightly from the location of the PSM 100 by placing aslot or notch 515 in the spacer surrounding the PSM 322 and 323 so thatair may vent out all around the PSM and not be obstructed in any fashionby the input whole blood that covers the PSM.

To operate the device, the input capillary tube 500 should be slightlyinclined with the body of the separation device slightly higher than thetip of the input capillary tube. The input is touched to a drop of bloodthat has formed on the surface of a finger previously punctured with alancet or needle. The capillary tube should continue to draw blood intoit until it reaches the end of the tube, which can be visually observed,or until it reaches the PSM itself, which is also visibly observable. Atthis time the device should be held upright to facilitate wicking of theblood across the whole upper surface of the PSM. Not all blood may leavethe input capillary tube initially, but may slowly move down as the PSMsaturates and plasma is extracted through the membrane.

If the input capillary tube is declined during filling, the blood in thetube may prematurely separate from the droplet on the finger and flow,by gravity, down the capillary tube. Attempting to add more blood maytrap a bubble, preventing further flow.

As the plasma collects in the pooling area 410, it will spread and movetoward an opening 450 which is the entrance of a capillary pipette 650that is inserted through the base of the device. Rather than just acapillary tube, the capillary pipette 650 is illustrated in FIGS. 3-4and 6-8 , which is a capillary tube 600 modified with a plastic sheathfor protection with finger gripping points 630, a squeeze bulb 670, avent in the squeeze bulb 610, and a fitting 620 and 625 that allows foreasy removal of the capillary pipette for dispensing the collectedplasma where it is needed. The capillary tube can be made of glass,plastic, metal or other material that is hydrophilic or can be renderedsuitably hydrophilic for the purpose of drawing plasma in by passivecapillary forces. The upper surface of the capillary 600 within thecapillary pipette 650 should be flush with the entrance of the hole orvia 450 in the plasma pooling areas 410.

Although some metering of the plasma volume takes place due to thegeometry of the pooling area, the exact volume of plasma collected intothe capillary pipette can be precisely controlled by the geometry of theinternal capillary 600 itself. Plasma enters this tube within thecapillary pipette and is held by capillary stop junctions at its inletand outlet. Air is vented out of the capillary through the small hole610 in the pipette's squeeze bulb 670. The pipette can be removed fromthe body of the device using the finger grips 630, not by holding on tothe squeeze bulb, and gently twisting the pipette while pulling it awayfrom the body of the device. Once separated, the pipette can be movedaround gently without fear of plasma exiting or dripping from it.

To dispense, the tip of the pipette is placed at the desired location ofdispensing, the bulb is squeezed, with the vent hole 610 covered, andthe plasma is then forced out of the internal capillary 600.

FIG. 3 illustrates a single, straight channel for the plasma poolingarea 410, and a friction fitting for the capillary pipette 620 and 625.The body of the plasma extraction device is either thermoformed orinjection molded and sealed or pinched round the edges including theedge of the PSM 100. FIG. 4 illustrates a laminated version of theplasma extraction device, with the plasma pooling area or channel 410formed in an adhesive layer 320 in the shape of a ‘U’. The capillarypipette 650 is held in place with a thin adhesive layer 625, and hasmore pronounced finger grips 630 to facilitate removal. A screw-type, orLuer fitting 620 can also be used to attach the capillary pipette to thebody of the device, in addition to friction or adhesive. Instead ofpinching or compressing the PSM 100 between the cover and base materialas shown in FIG. 3 , in FIG. 4 the PSM 100 is held within a cut-outsection of a spacer comprised of plastic 323 and adhesive 322 layers,and rests on the adhesive layer 320 that forms the channels of theplasma collection area.

In FIGS. 3 and 4 the internal alignment between the plasma pooling area410 and output capillary pipette 650 is critical to proper devicefunction. This alignment can easily be achieved using standard devicedesign and assembly methods. These include using robust, precision Luerlock fittings, or thin adhesive layers, with proper quality controls inplace.

Another important element in device usage is to ensure plasma extractionand filling of the capillary pipette is complete before removal of thecapillary pipette. This can be done by placing a contrasting agent ormark near the end of the capillary pipette that generates a visibledifference between filled and unfilled capillaries. This could be a verythin and readily soluble inert dye placed within the capillary itself.During manufacturing of the device the dye could be inserted a shortdistance at the distal end of the capillary and then blown out once ithas partially dried so that it does not clog or block flow of plasmafilling the pipette, but leaves a light colored film that disappearsonce plasma has filled the capillary. Another method may be to scratchthe inside of the distal end of the capillary with a diamond-tip bit, orsimilar device. The scratch marks would disappear when the capillary isfilled with plasma. Alternatively, the inside surface of the distal endof the capillary could be coated with a hydrophobic film so that it isnot filled. The difference between a filled and unfilled portion of thecapillary, when they are side by side, can be recognized. Finally, ifthe capillary is held vertically, a very small volume of plasma mayextend past the end of the capillary, showing that it is filled.

Direct Dispensing: In a previous example, the radius of the capillarywas suggested to be 150 μm. If the desired volume the capillary pipetteis to collect and dispense is 5 μL of plasma, then its required lengthcan be easily determined by the relationship between volume and lengthof a cylinder with a known radius, as illustrated by the equation:

$\begin{matrix}{{Length}_{c} = \frac{Vol}{\pi r_{c}^{2}}} & (8)\end{matrix}$

-   -   where: r_(c) Radius of the capillary    -   Vol Desired capillary collection volume

For the case cited, Vol=5 μL, r_(c)=150 μm, then Length_(c)=70.8 mm, or2.8 inches. In the design illustrated in FIG. 4 , this Length_(c)represents the length of the capillary 600 inside narrow diameterportion of the capillary pipette 650. The capillary can extend into thebulb region, but the minimum length of the entire capillary pipette willneed to be at least 70.8 mm. Such a long length of an unsupported glassmember begins to introduce fragility into the device design.

Consider further a case when a capillary tube of the same radius isused, but the desired collection volume is 10 μL rather than 5 μL. Thisrequires a capillary of double the previous length, or 141 mm. It ispossible the radius of the capillary tube can be increased to allow fora shorter capillary, using equation (8), in order to maintain a maximumcapillary length of 70.8 mm, r_(c) will need to be 212 μm to accommodatea volume of 10 μL. Fortunately this still satisfies the conditiondescribed by equation (7), using the same materials and geometry as usedpreviously, with an effective r_(p) of 150 μm. However, this requiresthe alignment of the output capillary to be much more precise in orderto ensure reliable operation, which may be beyond the manufacturingconstraints that allow a cost effective device to be produced.

An alternative solution to the very long capillary tube or highprecision manufacturing and assembly needs, is illustrated in FIG. 5 .In this design the collected plasma is dispensed directly from the bodyof the device, rather than collected and dispensed from a removablecapillary pipette.

The alignment challenges are much more relaxed because it is notintended for plasma to enter the squeeze bulb 770 or dispensing tip 870regions under capillary forces because the interface to these regionsare designed to represent capillary barriers which are not pushed pastduring plasma filling of the pooling or collection channel 410. Rather,positive pressure is applied by squeezing the bulb 770 to dispenseplasma out of the pooling area, through the tip 870 and into an externalreceiving area.

An input capillary cover or cap 501 is needed to ensure any remainingwhole blood is not ejected out through the input capillary 500 when thesqueeze bulb is actuated. The suspended or unsupported area of the PSM100, directly above the pooling area, may act as a type of movablediaphragm when the squeeze bulb is actuated. Care is needed to preventthis movement from causing leakage or ejection through the inputcapillary 500. The cap 501 would be put in place after the plasma hasfilled the plasma pooling area and before the squeeze bulb is actuated.

Aliquot Plasma: FIG. 6 illustrates a design where a single input volumecan be aliquoted into two output volumes. This provides anotheralternative to preventing excessively long output capillary pipettes,which can be accomplished by splitting the output into two differentcapillary pipettes 651 and 652, of either equal or unequal volumes.However, the primary advantage of this design is to enable dispensingtwo plasma volumes into two different destinations, but with only asingle input requirement.

This method requires the two pooling areas 411 and 412 to be distinctfrom each other, including two separate CPR-valves 201 and 202. However,they may share a common vent 330 because the plasma does not connect orspan across the vent in the design illustrated. Sharing a single channel(without a vent separating the two aliquots) and/or sharing a singleCPR-valve leads to irregular and non-repeatable behavior, and does notensure the two aliquots are divided repeatedly and reliably into thedesired volumes.

Plasma Flow Within the PSM: Many of the membranes developed for plasmaseparation were initially developed for lateral flow applications, suchas in lateral flow immuno-assays. Hence, the separated plasma canactually flow quite well within the lateral dimensions of the PSM.Referring to FIG. 4 , it has been observed on numerous occasions thatthe output capillary pipette 650 is able to completely fill before anyair enters into the plasma pooling channel 410 through the vent 330.This can only be explained by plasma flowing laterally from any point onthe PSM 100 and emptying into the channel 410 which feeds into theoutput capillary 600, rather than the pooling channel filling once andemptying once into the capillary.

This means that, at least in some cases, the plasma pooling channel 410under the PSM is not effective in metering the volume of plasma passeddownstream. Fortunately, the output is effectively metered by the outputcapillary 600. However, if the plasma pooling channel is not useful formetering, it can be eliminated or modified to serve other usefulfunctions. Three modifications that can be made include 1) eliminatingthe output venting duct to simplify manufacturing; 2) making the poolingchannel shorter to facilitate plasma reaching the output capillary morequickly; and, 3) making the adhesive layer thicker to allow for a largerdiameter output capillary to be used while still retaining sufficientcapillary forces needed to draw plasma into the output capillary.

Regarding a thicker adhesive layer, or greater distance between thebottom surface of the PSM and top surface of the base of the device, ifthis distance is too large it can become difficult for the meniscus toform between these two surfaces. However, assuming a meniscus does form,the larger space also makes the meniscus move more slowly forward. Thisis the reason the pooling channel can be shortened, so the more slowlyadvancing meniscus still reaches the output capillary within areasonably short period of time.

If the space between the two surfaces is greater, the effectivehydraulic radius discussed above with respect to equations (4)-(7) willbe larger, which means the allowable radius of the output capillary canbe larger, meaning its length shortened for the same metered volume.

Metering by the volume of the pooling channel under the PSM can still beachieved if the speed of filling of the output capillary tube, or otheroutput structure, exceeds the speed of lateral flow of the plasma tofill, or re-fill, the pooling channel. But, in the case of an automatic,stand-alone centrifuge replacement device, with an output capillarypipette, it is better for the output capillary to control metering dueto the three benefits described above with reference to FIG. 4 . Anexample of a device using these three benefits is illustrated in FIG. 7, where the normal metering plasma pooling channel 410 of earlierfigures is replaced with a shorter, deeper and non-metering plasmapooling channel 413, and the venting duct 330 of previous figures is nolonger used.

CPR-Valve Double Duty: Despite the three benefits described above, thedesired processing and collection volume of plasma may be far largerthan can be accommodated using thicker adhesives, shorter poolingchannels or other means of balancing the capillary force equationsdiscussed previously. According to equation (7), and using the samematerials and geometries mentioned in that case, if the output capillaryhas a radius that is larger than 3.7 times the effective hydraulicradius of the plasma pooling channel, then the interface between theoutput capillary and the plasma pooling channel represents a capillarystop junction and the plasma will not enter into the output capillary.

However, the whole point of this disclosure and function of theCPR-Valve or mechanism, is to not be limited by capillary stop junctionsor other capillary barriers that may exist in certain systems. As isillustrated in FIG. 8 , the CPR-Valve 200 can be designed to serve adual purpose, both to eliminate the breakthrough pressure of the PSM100, and to simultaneously overcome the capillary stop junction presentat the inlet of the output capillary 600.

Both the illustration in FIG. 8 , and further detail shown in FIG. 9 ,the CPR-Valve 200 is placed between the output capillary 600 and the PSM100, with a portion of the CPR-Valve extending past the output capillary600 to lie between the PSM 100 and base 310. The CPR-Valve firstovercomes the breakthrough pressure of the PSM, causes the meniscus ofthe pooling plasma to spread radially outward through the channels,spokes or arms of the plasma pooling area 413 extending from the centerand reaching out to the edges of the PSM, and at the same timeintroduces the plasma into the output capillary. As in the short channeldesign illustrated in FIG. 7 , this design relies on plasma flowinglaterally in the PSM before it empties into the collection channels. Thenumber of spokes or channels can be optimized depending on the desiredspeed of collection, surface area of the PSM, device fabrication,assembly and reliability issues.

Timing of the spreading plasma and its introduction into the outputcapillary is important. If the thickness of the adhesive 320, or channeldepth between the PSM 100 and base 310, is kept very small or thin suchas between 25-50 μm, and the width of each channel 413 is kept small,such as less than 1 mm, the meniscus will spread outward rapidly.Although a larger number of branches or spokes could increase the speedof filling the output capillary, it also increases the chance of onebranch not filling properly and trapping a bubble instead. If theCPR-Valve 200 introducing plasma into the output capillary 600 isslightly thicker, or denser than it is outside the capillary where itcontributes to overcoming the PSM breakthrough pressure, then it willtake a slightly longer time for the plasma to be introduced into theoutput capillary 600. It is important that sufficient plasma has passedinto the pooling space or branches 413 and extends to the pooling areavents 331, before the plasma is drawn into the output capillary 600, sothe output capillary does not draw an air bubble into it rather thanplasma. In this case the vents 331 shown in FIG. 8 are not used tointroduce air into the pooling area as plasma enters the outputcapillary, as is the case for vents 330 shown in previous figures, butrather to vent air out of the pooling channels 413 as they fill,beginning at the center of the device where the CPR-Valve 200 islocated, and expanding radially outward. If plasma reaches the vents331, air will not re-enter through the vent into the pooling channels413 because the adhesive layer 320 is so thin, a strong capillaryjunction exists at the point of the vent 331, and air will not enter. Inthis case, the total volume of plasma in the pooling channels 413 willcontribute to the dead volume of the system because it cannot becollected into the output capillary.

Initially plasma is drawn into the output capillary by capillary force.But, depending on the geometry and volume of the output capillary, atsome point the weight of the liquid column (if the device is heldupright) will begin to act as a suction source that will draw an airbubble into the capillary 600 if plasma in the channels 413 have not yetreached the output vents 331. This appears to become a factor when theliquid column height in the output capillary 600 approaches 20 mm orgreater. To prevent this from happening, the pooling space or channelsunder the PSM should be filled with plasma rapidly without trappingbubbles. Alternatively, the device could be placed on its side tominimize any suction pressure that is generated by the column of plasmain the output capillary. In this case the squeeze bulb 670 should beresting on the support surface, such as a table, with the inlet of thecapillary, within the device, slightly higher in elevation than thesqueeze bulb. This will allow capillary forces to continue to functionproperly and gravitation forces to only be slightly additive, but weakerthan if the device is in a vertical position.

In the case where the CPR-Valve 200 is between the output capillary 600and the PSM 100, if the soluble material comprising the CPR-Valve isdyed, it can be observed that the material is first drawn radiallyoutward by the radially expanding meniscus, but then is it drawn inwardby the flow of plasma entering the output capillary. In contrast to theprevious design where most or all of the CPR-Valve material can beexcluded from the collected plasma volume, in the current design FIGS. 8and 9 the CPR-Valve material, either entirely or in large part, will bedrawn into the output capillary rather than cut-off from collection, ashas been described previously. In this case it is of more importance forthe material comprising the CPR-Valve to not be an interferent in anydownstream process, such as in biomarker detection, than in the casewhen all or the majority of dissolved CPR material is excluded fromcollection.

Integrated Reagent: As has been mentioned, in the design illustrated inFIGS. 8 and 9 , all or the majority of the material comprising theCPR-Valve 200 will be collected by the output capillary 600. Thus, thematerial should not interfere in downstream processes, and since it isreliably and repeatedly passed downstream, the CPR-Valve can becomprised of materials important to downstream processing. This isillustrated in FIG. 10 where a reagent disk 250 is located between theCPR-Valve 200 and the output capillary 600.

Instead of being 2 different structures or materials, the CPR-Valve 200and reagent disk 250 can also be the same structure and material,depending on the needs of the system. In this case the plasma separationdevice is not necessarily an independent stand-alone centrifugereplacement tool but, due to the presence of a specific reagent that isclosely associated with a specific application or diagnostic test wherethe reagent is used, it becomes an integral component of a specificdiagnostic application or test. The reagent 250 should also be presentin excess of the minimum needs of the application, to ensure adequatereagent is present in the downstream system if not all of the reagent iscollected in the output capillary 600. A common example of this isconjugate reagent being present in excess for downstream lateral flowdevices.

In many diagnostic applications reagents are present in the form oflyophilized beads or pellets. It is possible they can be in the form ofa disk that is in contact with the CPR-Valve 200. It is also possiblethe reagent can be used as a CPR-Valve. The primary requirements of aCPR-Valve are that is possesses high capillarity in dry form, to drawliquid out of the PSM, that it facilitates the generation of a meniscusbetween the PSM and base of the device, and that it is readily solublein the filtrate or plasma without increasing the filtrate or plasma'sviscosity to any great degree. Some characteristics of lyophilizedmaterials may not allow it to be used as a CPR-Valve, and may complicatethe introduction of liquid into the output capillary, such as very rapidif not explosive dissolution or disintegration in the presence ofliquid, which may complicate meniscus formation or inject bubbles intothe output capillary. If this is the case the reagent 250 could beplaced near the CPR-Valve 200 and/or output capillary 600, rather thanused instead of the CPR-Valve or in contact with the output capillary.Even in this condition it is very likely the majority of the reagentwill be collected into the output capillary during the separation andcollection process.

Surface Area, Hematocrit and Input and Output Volumes: Because most PSMsare asymmetric size exclusion filters, ranging from large pore size atthe top to smaller pore size at the bottom, if sufficient blood ispassed through them, they will eventually clog with cells preventingfurther plasma separation or extraction. Because of this, commercialPSMs are often rated in volume of input blood per square centimeter ofsurface area of the filter, or membrane.

For example, the GR grade of the Pall Vivid PSM states 40-50 μL of wholeblood input per square centimeter of membrane surface area. However, inthe laminated designs illustrated in FIGS. 2, 4-8 , a large portion ofthe bottom surface of the PSM is blocked by adhesive 320, which is usedto support the membrane and to define the geometry of the plasma poolingarea 410, 411, 412 or 413. Unfortunately, however, this does not meanthat the corresponding upper surface of the PSM does not need to becovered, which would reduce the total volume of whole blood required.Experimental analysis has shown that the entire upper surface area andtotal depth of the PSM must be saturated with the input whole blood inorder for the devices to function properly. This reduces the efficiencyof separation. In a thermoformed or injection molded design, however, itis possible to pinch or squeeze-off a portion of the upper surface areaand corresponding depth or thickness of the PSM, which would allow anupper surface area to approximately equal the top of the plasma poolingarea. This would increase the efficiency of the system.

Another factor effecting potential plasma volume is the hematocrit ofthe input whole blood sample. Hematocrit is the ratio of whole bloodvolume to packed cellular components of the blood. A low hematocritrepresents a low cellular component of blood and larger liquid, orplasma component. A high hematocrit represents a higher cellularcomponent and lower liquid or plasma component. Typical physiologicalranges of hematocrit variability among healthy individuals is between38-55%, meaning a maximum volume of 62 μL of plasma could be extractedfrom an individual with a hematocrit of 38%, if the starting whole bloodvolume were 100 μL, but only 50 μL if their hematocrit were 50%. Inaddition, unhealthy individuals, or people acclimated to extremeconditions, such as high altitude, infants, and other sub-groups mayhave hematocrit ranges much broader than this. Hematocrit variability,if not accounted for, can represent a significant variation and sourceof error in blood-based diagnostics, where the measurement of biomarkersand their physiological significance is measured in units of plasma orserum and not whole blood.

In order to ensure proper device function, and taking into account deadvolume, surface area specifications, blood or plasma retained with thePSM or overall efficiency in separation of the PSM, and potentialhematocrit variability, a device that specifies a fixed and accurateplasma extraction, metering and dispensing capability will, by itsnature, be inefficient in its ability to collect the total volume ofplasma available in a sample.

Depending on the overall design of the devices illustrated in thefigures, and depending on whether they are molded, thermoformed orlaminated, and depending on the allowable range of hematocrit that canbe accommodated for, the devices disclosed in this filing are capable ofextracting approximately 1 μL of plasma from every 3 to 6 μL of wholeblood input.

Another embodiment of the technology is illustrated in FIG. 11 , wherethe efficiency of the system can be improved by having an extension 415of the plasma pooling area 410 that extends past the boundary of the PSM100. The depth or thickness of the pooling area cannot be too thickbecause of the difficulty in establishing a meniscus between the upperand lower surfaces, and because of the need for capillary forces to helpdrive the meniscus, which capillary forces may become too weak if thethickness or depth of the pooling area 410 becomes too large, such asgreater than 300 μm. However, a microchannel or extension 415 with ahigher capillary force than the pooling area 410 directly under the PSM100 can serve to draw some of the plasma out from under the PSM, whichis replaced by more plasma passing through the PSM. The interface to theoutput capillary 600 could then connect with this extension 415.However, in this case the capillarity of the output capillary 600 mustbe higher than the capillarity of the extension 415, rather than thecapillarity of the pooling area 410 under the PSM 100.

Input Volume Metering: As has been mentioned, depending on a capillarytube's geometry (e.g., inner-diameter) and composition (e.g., glass orplastic), and the liquid that it holds (e.g., blood) which define itscapillarity, gravity can have an effect on the position or movement ofliquid inside. For example, a small amount of plasma can extend past theend of the output capillary, signaling it is full. This is due to theweight of the liquid pushing on the meniscus, and the fact that it therim and edges of the capillary may still have strong capillary forces,or high surface energy, allowing the plasma to migrate slowly down andout when, theoretically, the abrupt end of the capillary shouldrepresent a stop junction which should prevent any further movement ofthe plasma. This creeping outward is slowed or stopped when thecapillary is held in a horizontal position, indicating gravity has aneffect on the liquid in the capillary.

Factors that influence the spread of liquid out of the capillary includethe height of the column of liquid, the ID (inner-diameter) of thecapillary, its density which, together with ID and column heightindicate the mass of the liquid, the thickness of the glass capillarywalls and the angle of the capillary with respect to gravity.

For example, it has been observed that, when held vertically, the bloodwithin a 1 mm ID glass hematocrit tube will extend past the bottom ofthe tube by about 1 mm if the column of blood within the tube is atleast 30 mm in height. But, it will only extend past the bottom of thetube by about 0.5 mm if the column height is reduced to about 25 mm.Also, the edges of the extension of the meniscus past the end of thetube can be seen to migrate from being constrained to the inner walls ofthe capillary to move to the outer edge of the capillary when the columnis higher.

If a first capillary tube, where the sample is introduced, is held in aslightly inclined position such that the distal end is slightly elevatedabove the proximal end, between 0.5 to 30 degrees up from thehorizontal, the sample should fill the tube automatically by capillaryforce and stop at the distal end. If the distal end is enclosed inanother capillary tube that has an ID (inner-diameter) that tightly fitswith the OD (outer diameter) of the first tube, the capillary stopjunction at the distal end of the first tube should still stop the flowof liquid filling it at its proximal end. Even though it is enclosed ina second tube, the ID of the flow channel still increases sufficientlyto generate a capillary stop junction.

If, however, once the first tube fills to its end point, both tubes areheld upright or vertical, depending on the length of the first tube, theweight of liquid inside it can be sufficient to draw it past its stopjunction until the extended meniscus contacts the ID of the second tube,causing it to move forward, eliminating the stop junction, and allowingliquid to move forward. In this case the weight of the liquid in thecolumn breaks the capillary stop junctions at both the inlet and outletof the first capillary tube.

This situation is illustrated in FIG. 12 a and b where the distalcapillary stop junction 580 of the first capillary tube 500 can be usedto precisely meter the input volume of liquid (blood) 900 that is loadedinto the first tube as it is in its loading position, with the distalend slightly elevated above the input. However, when the system is heldupright the weight of the sample 900 can push it past this stop junction580 and into the second tube 590, which leads to the downstream devicewhich must have appropriate properties to draw in the metered inputvolume, such as by having sufficient capillarity to draw liquid out ofthe first 500 and second 590 capillary tubes, in this case a PSM 100.

This design can more precisely meter the input of a system where moreprecise metering is important. In the design mentioned previously only aminimum volume of input is ensured, but the maximum was not controlled.In this new design the minimum and maximum are controlled, by filling tothe stop junction 580 of the first capillary 500, which can be visiblyobserved. An example of when input metering is more critical is when theplasma extraction device disclosed herein is optimized for function as ahematocrit measurement device, where both input and output are measured.

Although a few exemplary embodiments of this invention have beendescribed, those skilled in the art will readily appreciate that manymodifications are possible in the exemplary embodiments withoutmaterially departing from the novel teachings and advantages of thisinvention. Accordingly, all such modifications are intended to beincluded within the scope of this invention as defined in the claims.Therefore, it is to be understood that the foregoing is illustrative ofthe present invention and is not to be construed as limited to thespecific embodiments disclosed, and that modifications to the disclosedembodiments, as well as other embodiments, are intended to be includedwithin the scope of the appended claims. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

1. (canceled)
 2. A device for collecting a metered volume of liquid froma liquid filtrate sample, the device comprising: at least one filtrationmembrane; at least one filtrate collection structure to collect liquiddrawn through the filtration membrane, the liquid drawn through thefiltration membrane constituting the liquid filtrate sample; at leastone liquid collection structure enclosing a known volume and possessingsufficient capillarity to fill with and thereby meter a volume of liquiddrawn from the liquid filtrate sample in the filtrate collectionstructure; and an indicator region at a distal end of the liquidcollection structure, the indicator region configured to visiblyindicate when the liquid collection structure has filled with the liquiddrawn from the liquid filtrate sample.
 3. The device of claim 2, whereinthe indicator region comprises a soluble colored dye or contrastingagent deposited on an inside surface of the liquid collection structure,wherein the dye or contrasting agent dissolves in the liquid drawn fromthe liquid filtrate sample once said liquid fills the liquid collectionstructure.
 4. The device of claim 2, wherein the indicator regioncomprises one or more scratches or marks on an inside surface of theliquid collection structure, wherein the scratches or marks visuallydisappear once the liquid drawn from the liquid filtrate sample fillsthe liquid collection structure.
 5. The device of claim 2, wherein theindicator region comprises a hydrophobic film on an inside surface of adistal portion of the liquid collection structure that prevents thedistal portion of the liquid collection structure from filling with theliquid drawn from the liquid filtrate sample, thereby providing avisible contrast between filled and unfilled portions of the liquidcollection structure once the liquid drawn from the liquid filtratesample fills the liquid collection structure up to and excluding thedistal portion.
 6. The device of claim 2, wherein the liquid collectionstructure comprises a capillary tube.
 7. The device of claim 6, whereinthe liquid collection structure further comprises a fitting, thecapillary tube being removably attached to the filtrate collectionstructure via the fitting.
 8. The device of claim 7, wherein the liquidcollection structure comprises a squeeze bulb for dispensing the meteredvolume of liquid drawn from the liquid filtrate sample following removalof the capillary tube from the filtrate collection structure.
 9. Thedevice of claim 8, wherein the squeeze bulb comprises a vent.
 10. Thedevice of claim 8, wherein the liquid collection structure comprises aplastic sheath surrounding the capillary tube, the plastic sheathdefining finger gripping points on the capillary tube.
 11. The device ofclaim 2, wherein the at least one filtration membrane comprises a plasmaseparation membrane.
 12. The device of claim 2, wherein the filtratecollection structure comprises a base material and walls that, togetherwith the at least one filtration membrane, enclose a filtrate poolingspace.
 13. The device of claim 2, wherein the at least one liquidcollection structure comprises a base material and walls that, togetherwith the at least one filtration membrane, enclose first and secondfiltrate pooling spaces, and wherein the at least one liquid collectionstructure comprises a first liquid collection structure configured tofill with and meter a first volume of liquid drawn from the firstfiltrate pooling space and a second liquid collection structureconfigured to fill with and meter a second volume of liquid drawn fromthe second filtrate pooling space.
 14. The device of claim 2, furthercomprising an input metering capillary configured to deliver a minimumvolume of liquid sample to an upstream surface of the at least onefiltration membrane.
 15. The device of claim 14, further comprising acover or cap associated with the input metering capillary that is put onthe input metering capillary once the liquid collection structure hasfilled with the liquid drawn from the liquid filtrate sample.
 16. Adevice for collecting a metered volume of liquid, the device comprising:a liquid collection structure enclosing a known volume and configured tofill with and thereby meter a liquid; and an indicator region at adistal end of the liquid collection structure, the indicator regionconfigured to visibly indicate when the liquid collection structure hasfilled with the liquid.
 17. The device of claim 16, wherein theindicator region comprises a soluble colored dye or contrasting agentdeposited on an inside surface of the liquid collection structure,wherein the dye or contrasting agent dissolves in the liquid once saidliquid fills the liquid collection structure.
 18. The device of claim16, wherein the indicator region comprises one or more scratches ormarks on an inside surface of the liquid collection structure, whereinthe scratches or marks visually disappear once the liquid fills theliquid collection structure.
 19. The device of claim 16, wherein theindicator region comprises a hydrophobic film on an inside surface of adistal portion of the liquid collection structure that prevents thedistal portion of the liquid collection structure from filling with theliquid, thereby providing a visible contrast between filled and unfilledportions of the liquid collection structure once the liquid fills theliquid collection structure up to and excluding the distal portion. 20.The device of claim 16, wherein the liquid collection structurecomprises a capillary tube.
 21. The device of claim 20, wherein theliquid collection structure further comprises a squeeze bulb fordispensing the metered volume of liquid.